Muscarinic Effects on Cellular Functions in Cultured Human Ciliary Muscle Cells Shun Matsumoto, * Thomas Yorio, * Louis DeSantis,] and Iok-Hou Purpose. To characterize the pharmacology of the carbachol-induced changes of phospholi- pase C (PLC) activity and intracellular calcium concentration ([Ca2+]j) in cultured human ciliary muscle cells. Methods. Changes in PLC activity of cultured human ciliary muscle cells were determined by production of inositol phosphates. Single-cell dynamic fluorescence ratio imaging was used to determine [Ca2+];. Results. Carbachol, oxotremorine-M, aceclidine, and pilocarpine stimulated PLC with mean ECsoS of 20, 8, 17, and 2 (JM, respectively. The effect of carbachol on PLC was partially suppressed by extracellular Ca2+ depletion. This muscarinic effect was blocked by muscarinic antagonists, such as atropine (apparent pKj = 9.12, nonselective for muscarinic receptor subtypes), pirenzepine (pKj = 6.76, selective for the Mi receptor subtype), 4DAMP (pKi = 9.25, selective for the M, and M3 subtypes), and fHHSiD (pK< = 7.77, selective for the M3 subtype). In [Ca2+]j experiments, carbachol increased [Ca2+]i transients in human ciliary muscle cells in a dose-dependent manner with a mean EC50 of 7 fjM. 4DAMP was approxi- mately 100 times more potent than pirenzepine in the inhibition of the carbachol-induced [Ca2+]j increase. [Ca2+]j oscillations were observed after carbachol stimulation and persisted after extracellular Ca2+ depletion. Conclusions. Muscarinic agonists activate PLC and increase [Ca2+]i in cultured human ciliary muscle cells through an M3-like muscarinic receptor subtype. Invest Ophthalmol Vis Sci. 1994; 35:3732-3738. L opical application of muscarinic agonists onto the types, named mi to m5, were discovered, mi, m2, and eye lowers intraocular pressure, presumably because m3 correspond to, respectively, the Mb M2, and M3 of the activation of muscarinic acetylcholine receptors subtypes defined by affinity profiles of selective musca- (mAChR), which in turn induce contraction of ciliary rinic antagonists.5 Because both pilocarpine and car- muscles and increase aqueous humor outflow.12 How- bachol are nonselective among the five receptor sub- ever, in addition to their action on aqueous dynamics, types, it is not clear which receptor subtype or subtypes muscarinic agonists, such as carbachol and pilocar- are responsible for the ocular actions of the muscarin- pine, also have ocular side effects. Their common ad- ics. Therefore, it is possible that their ocular hypoten- verse effects include miosis (due to the contraction of sive effects and their side effects are mediated by dif- the iris sphincter muscle), accommodation (change ferent subtypes and, consequently, that appropriate of lens curvature secondary to ciliary muscle contrac- subtype-specific agonists theoretically could lower in- tion), and browache (supposedly due to severe con- traocular pressure with reduced ocular side effects. traction or spasm of the ciliary muscles) .3>4 Indeed, current evidence indicates that one can sepa- Recently, messenger RNAs of five mAChR sub- rate the therapeutic from the untoward effects of the muscarinics. Aceclidine, another muscarinic agonist, is equipotent to pilocarpine in lowering intraocular From the *Department of Pharmacology, North Texas Eye Research Institute, pressure in human subjects, but it is more potent as University of North Texas Health Science Center at Fort Wmth, and ^Glaucoma 6 9 Research, Alcon Laboratories, Fort Wmth, Texas. a miotic and does not cause much accommodation. " Supported in part by a grant from Alcon Laboratories. In monkeys, aceclidine similarly increases the outflow Submitted for publication September 3, 1993; revised February 17, 1994; accepted April 25, 1994. facility of aqueous humor with minimal accommoda- Proprietary interest category: E, C5. tive changes.10 Additionally, the pilocarpine-induced Reprint requests: Shim Matsumoto, M.D., Department of Ophthalmology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunhyo-ku, Tokyo 113, Japan. increase in outflow facility in the monkey can be re- Investigative Ophthalmology & Visual Science, September 1994, Vol. 35, No. 10 3732 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/28/2021 Muscarinic Effects on Ciliary Muscle Cells 3733 versed by atropine in two phases, a fast and a slow dium was removed and each well was rinsed with 4X1 phase, whereas the reversal of accommodation shows ml DMEM:nutrient mixture F12 (1:1), supplemented only the fast phase.11 This dissociation of the musca- with 10 mM LiCl (Mallinckrodt, Paris, KY) at room rinic effects agrees with the hypothesis that different temperature. In some experiments, Ca2+ in the me- effects are mediated by different receptor subtypes. dium was chelated with additional ethyleneglycol bis An initial step to test this hypothesis is to identify (2-aminoethylether) tetraacetic acid (EGTA; Sigma, St the receptor subtypes located in the different ocular Louis, MO), forming an EGTA-Ca2+ buffer system (pH tissues. Several published studies attempted to address = 7.4) .20 The cells were incubated with 1 ml of the this issue using receptor binding or techniques of mo- same solution for 10 minutes before 10 (A of agonists lecular biology. In the human iris sphincter, the m3/ were added to the indicated wells. Alternatively, antag- M3 subtype was shown to be the most prominent onists were added at this time and 10 fi\ of carbachol 12 14 mAChR. " The messenger RNA of the m3 receptor (final concentration = 100 /iM) was added 12 minutes was also detected in cultured human ciliary muscle later. After an incubation period of 1 hour, the assay cells and in postmortem tissues.15"17 Recently, based was stopped by the removal of the medium, and 0.5 on results from receptor binding assays, WoldeMussie ml of perchloric acid (Baker, Phillipsburg, NJ) was 12 et al reported that an M3-like mAChR subtype is pres- added. Samples were then incubated on ice for 10 ent in cultured human ciliary muscle cells, and it was minutes. The perchloric acid was later removed by suggested that this receptor may be involved in the extraction with 2 ml of 1:1 (vol/vol) 1,1,2-trichloro- carbachol-induced activation of phosphoinositide me- trifluoro-ethane:tri-n-octylamine (Sigma) mixture. tabolism. Because studies that use ligand binding or The resulting aqueous layer was loaded onto anion- Northern blot analysis can only demonstrate the pres- exchange columns (AG 1-X8 anion exchange resin ence and the cellular concentration of the receptor in formate form, 1 ml; BioRad, Hercules, CA). The protein and the messenger RNA, they do not directly columns were washed with 10 ml of water and 7 ml define the relative importance of the receptor sub- of 50-mM ammonium formate (Sigma). The bound types in cell functions. To expand these studies fur- inositol phosphates were then eluted with 2X2 ml ther, we used cell functional assays (phospholipase C of 1.2 M ammonium formate-0.1 M formic acid activity and intracellular calcium concentration) to try (Sigma). The radioactivity in the eluates was counted to elucidate the functional involvement of muscarinic with a /3-scintillation counter. In recent experiments, receptor subtypes in the cultured human ciliary mus- the assay procedures were simplified such that the cle cells. reaction was terminated by replacing the cell medium with 1 ml of 0.1-M formic acid instead of perchloric acid. The cell lysate was then loaded directly onto the MATERIALS AND METHODS anion-exchange column and eluted, and radioactivity was measured as described. The two assay procedures Cells generated similar results (data not shown). PLC activ- The human ciliary muscle cell strain established by ity of the cells was estimated from the accumulation Tamm et al18 was used. The cells were cultured at of radioactive inositol phosphates. 37°C in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY), supplemented with 10% fetal calf Intracellular [Ca2+] Measurement serum (Hyclone, Logan, UT), 4 mM L-glutamine Human ciliary muscle cells were grown on coverslips (Gibco), and 50 Atg/ml gentamicin (Gibco) in humidi- (#0; Biophysica Technologies, Baltimore, MD) in 6- fied air containing 5% CO2. Upon confluence, the well plates (Costar). The culture medium was changed cells were subcultured by trypsinization using 0.05% to serum-free DMEM 24 hours before the calcium con- trypsin-0.53 mM ethylenediamine-tetraacetic acid centration measurement. On the day of the measure- (Gibco). Cells of passages 10 to 14 were used in subse- ment, the cells were incubated for 60 minutes in a quent experiments. HEPES buffer (125 mM NaCl, 5 mM KC1, 1.8 mM CaCl , 2 mM MgCl , 0.5 mM NaH PO , 5 mM Phospholipase C Assay 2 2 2 4 NaHCO3,10 mM HEPES, 10 mM glucose, 0.1% bovine Phospholipase C activity was assayed according to a serum albumin; pH 7.2) containing 5 fiM of a calcium- procedure modified from that described by Downes fluorescent dye, fura-2 acetoxymethyl ester (Fura-2/ and Michell.19 Human ciliary muscle cells grown in AM; Molecular Probes, Eugene, OR). After the incu- 24-well plates (7 X 105 to 8 X 105 cells per well; Costar, bation, the coverslip was rinsed twice with the same Cambridge, MA) were incubated with myo-[2-3H]-ino- HEPES buffer and mounted in a chamber on the stage sitol (specific activity 370 to 740 GBq/mmol; Amers- of a Nikon Diaphot microscope (Tokyo, Japan). The ham, Arlington Heights, IL) at 5 /t/Ci/ml per well in chamber was filled with 3 ml of the HEPES buffer serum-free Dulbecco's modified Eagle's medium and kept at 37°C during the experiment. Intracellular (DMEM) for 2 days. On the day of the assay, the me- fluorescence intensity of 510-nm emission wavelength, Downloaded from iovs.arvojournals.org on 09/28/2021 3734 Investigative Ophthalmology 8c Visual Science, September 1994, Vol.